Physiological studies have been aided by the use of intracellular indicators. Indicators for intracellular calcium and pH are used in monitoring physiological and pathophysiological properties of isolated cardiac myocytes and intact cardiac tissue. This project developed a novel time resolved system for cytosolic pH measurements using the recently synthesized intracellular pH indicator, SNARF-1, seminaphthorhodalfluor, with simultaneous measurements of cell length. The emission spectrum of SNARF-1 contains two-well separated emission peaks at 590 and 640 nm. This feature allows the indicator to be used in the single excitation, dual emission, ratio mode; analogous to the calcium indicator, INDO-1. SNARF-1 is available in both the free acid form and as a cell permeant acetoxymethyl ester. We have found that isolated cardiac myocytes are easily loaded with ester, and have the following characteristics: 1) a consistent intracellular calibration can be obtained, 2) the contractile properties are essentially unchanged in the presence of the indicator, 3) the indicator is present primarily in the cytosol (95% to 10010/0) with virtually no partitioning into the mitochondria, 4) the indicator is retained for several hours at room temperature, and 5) steady-state pH and transient changes in pH are easily monitored. Changes in pH can be monitored during important physiological and pathophysiological perturbations. The initial applications of the SNARF-1 system include 1) pH regulation and changes in contractile state during anoxia, acidosis, and anesthesia and 2) receptor mediated changes in contractile state.